Mycobacteria are unicellular, aerobic, Gram-positive bacteria. Typically, mycobacteria have a thick hydrophobic cell wall and lack an outer cell membrane. Infections caused by mycobacteria can be active within a host, or latent and asymptomatic. The emergence of multi-drug resistant strains, the need for prolonged antibacterial therapy, and poor patient compliance, has made treatment of mycobacterial infections difficult, particularly in developing nations. The emergence of multidrug resistant (MDR) strains of M. tuberculosis, in particular, has made diagnosis and treatment of TB a high priority in developing African populations.
The primary consequence of mycobacterial infection (and particularly, infection by one or more species of Mycobacterium genus) in humans is tuberculosis (TB), a contagious infection caused by members of the “M. tuberculosis complex,” which include, e.g., pathogenic strains of the species M. tuberculosis, M. bovis, M. africanum, M. microti, M. cannetti, M. caprae and M. pinnipedi. TB typically attacks the lungs in mammalian hosts, but can also spread to other organs and regions of the body including, for example, bone, joints, kidneys, and the abdomen, etc. Members of the M. tuberculosis complex are closely related genetically, and possess highly-conserved 16S rRNA sequences across the genus.
TB can be acquired by breathing in air droplets from a cough or sneeze of an infected person. Symptoms of an active tubercular infection can include chronic cough (typically with blood-tinged sputum), fever, nocturnal hyperhidrosis, chronic fatigue, pallor, weight loss, and cachectic wasting (“consumption”). Other symptoms can include breathing difficulties, thoracic pain and wheezing (“Pulmonary Tuberculosis,” PubMed Health). If an inhaled tubercle bacillus settles in a lung alveolus, infection occurs, followed by alveolocapillary dilation, and endothelial cell swelling. Alveolitis results with intracellular replication of the tubercle bacilli, and an influx of polymorphonuclear leukocytes to the alveoli. The organisms then spread through the lymph system to the circulatory system, and then throughout the body.
Although M. tuberculosis infects less than 200,000 people annually in the United States, according to the World Health Organization (WHO) nearly two billion people worldwide may be infected, 90% of whom can remain asymptomatic for years following infection. Left untreated, TB is fatal in >50% of the infected population, and in disseminated forms of the disease, the mortality rate approaches 90%.
Because of the chronic and debilitating persistence of TB infection, co-infection with one or more secondary pathogens, including in particular, human immunodeficiency virus (HIV), is also widespread. In 2007, there were at least 1.37 million cases of HIV-positive TB, concentrated primarily in emerging populations where diagnosis and treatment are often limited, ineffective, and/or cost-prohibitive.
Conventional diagnosis of a TB infection typically relies on a combination of physical examination (e.g., chronic persistent cough, enlarged or tender lymph nodes, pleural effusion, unusual breath sounds, and, in later stages of the disease, characteristic “clubbing” of the fingers or toes) and diagnostic testing (e.g., sputum examination, microbial culture and nucleic acid testing of specimens, bronchoscopy, CT scan or X-ray of the chest, pulmonary biopsy, thoracentesis, interferon-γ (gamma) blood test, and tuberculin skin test).
The Mantoux tuberculin skin test, or purified protein derivative (PPD) skin test, is performed by intradermally injecting about 0.1 mL of tuberculin PPD into the inner surface of the forearm. Tuberculin PPD is a precipitate of non-species-specific molecules obtained from filtrates of sterilized, concentrated TB cultures. Immune reaction by the patient to PPD is measured within 48 to 72 hours of injection, as millimeters of induration (i.e., a palpable raised hardened area on the skin), which is dependent on the individual's risk factors for acquiring the disease. Both false-positive and false-negative results are fairly common, especially amongst those infected with either non-TB or TB mycobacteria, or a viral illness such as measles or chicken pox, or those previously vaccinated with BCG (Bacille Calmette-Guérin) or live-virus (“Tuberculin Skin Testing for TB,” Centers for Disease Control and Prevention).
Acid-fastness is a physical property of some bacterial species that refers to their resistance to decolorization by acids during microscopic staining procedures. In the most common of these procedures, the Ziehl-Neelsen test, the specimen is spread onto a microscope slide, exposed to particular dyes, and then decolorized with a dilute acid or alcohol. Because of the high mycolic acid content of mycobacterial cell walls, these so-called “acid-fast” organisms resist destaining, thus a smear-positive result is presumptively indicative of the presence of tubercle bacilli. Because other non-mycobacterial species may also appear acid-fast in this test, however, sensitivities of less than 50% have been reported when using acid-fastness as an identification criterion. Culture of the specimen and further biochemical testing are therefore required to definitively confirm the presence of TB.
Both the tuberculin skin test and the smear tests are used as screening methods and are not usually determinative of a TB infection, therefore even those individuals with smear-negative results can be further tested for TB, depending upon the individual's risk factors and availability of testing. M. tuberculosis detection using the GeneXpert® (Cepheid, Calif., USA) platform has been widely implemented throughout Africa and appears to detect a high percentage of smear negative cases. However, this also has limitations that include reduced sensitivity compared to other nucleic acid approaches, increased platform and testing costs and constraints for use in remote and point of care settings.
The “standard” of TB diagnostics, cell culturing of mycobacterial organisms, is difficult, due in part to their long generation times, i.e., twenty-four hours for M. tuberculosis. In addition, mycobacteria are typically present at low levels in infected individuals. Culturing from a clinical specimen can therefore take anywhere between four to eight weeks, during which time a patient may become seriously ill and contagious to others. In addition, cell culturing requires the collection, transport and maintenance of viable mycobacterial organisms in a sample until such time as the sample can be analyzed in a lab setting. In countries where TB is prevalent, and health care is minimal, this may not be an option, thus increasing the risk of spreading infection.
Interferon-γ tests, such as QuantiFERON® TB Gold (Cellestis Limited, Victoria, Australia), measure the amount of the cytokine, interferon-γ (IFN-γ) (a component of cell-mediated immune reactivity to the M. tuberculosis complex) and can detect both latent and active tuberculosis infections. Heparinized whole blood obtained from a person suspected of TB infection is incubated for 16-24 hours with ESAT-6 and CFP-10, two synthetic proteins derived from M. tuberculosis, and control antigens. The level of IFN-γ produced by the lymphocytes upon recognizing the synthetic proteins is measured, and results are unaffected by previous BCG vaccination or cross-reactivity with other mycobacteria. Results of the IFN-γ test can be later confirmed by standard culture methods, if necessary. Unfortunately for regions with limited access to medical care, the whole blood must be analyzed within 12 hours of obtaining the sample, and the effectiveness of the test has not been analyzed on patients with other medical conditions such as HIV, AIDS, diabetes, silicosis, chronic renal failure, hematological disorders, individuals that have been treated for TB infection, nor has it been tested on pregnant individuals or minors (“Clinicians Guide to QuantiFERON®-TB Gold,” Cellestis). Other non-culture methods such as radioimmunoassays, latex agglutination, and enzyme-linked immunosorbent assays (ELISAs) have been used with limited degrees of success to confirm the presence of tubercle bacilli in biological samples.
Nucleic acid amplification testing for TB includes the use of standard polymerase chain reaction (PCR) techniques to detect mycobacterial DNA in patient specimens, nucleic acid probes to identify mycobacteria in culture, restriction fragment length polymorphism (RFLP) analysis to compare different strains of TB for epidemiological studies, and genetic-based susceptibility testing to identify drug-resistant strains of mycobacteria. The complete genome of M. tuberculosis has been sequenced and published; currently two nucleic acid amplification-based tests for TB have been approved for use in the United States by the Food and Drug Administration (FDA). The first, known as the “Enhanced Amplified Mycobacterium Tuberculosis Direct Test” (E-MTD, Gen-Probe, San Diego, Calif., USA), is approved for detection of M. tuberculosis complex bacteria in acid-fast bacilli in both smear-positive and smear-negative respiratory specimens from patients suspected of having TB. The E-MTD test combines isothermal transcription-mediated amplification of a portion of the 16S rRNA with a detection method that uses a hybridization probe specific for M. tuberculosis complex bacteria. The second, known as the AMPLICOR® Mycobacterium tuberculosis Test (AMPLICOR®, Roche Diagnostics, Basel, Switzerland), has been approved for the detection of M. tuberculosis complex bacteria only in smear-positive respiratory specimens from patients suspected of having TB. This test uses PCR to amplify a portion of the 16S rRNA gene that contains a sequence that hybridizes with an oligonucleotide probe specific for M. tuberculosis complex bacteria. (“Report of an Expert Consultation on the Uses of Nucleic Acid Amplification Tests for the Diagnosis of Tuberculosis,” Centers for Disease Control and Prevention).
Results have indicated that the sensitivity and specificity of these tests tends to vary depending on geographical location and risk factors. In addition, these techniques require complex laboratory conditions and equipment to be performed, thus reducing the speed and sensitivity of the test. For these and other reasons, there remains a need in the art for reliable and accurate methods for detection of Mycobacterial pathogens in clinical samples, and in particular, methods for rapidly identifying such pathogens in field applications, remote locations, and in developing countries where conventional laboratories are lacking, and financial resources are limited. In particular, compositions for the safe collection, handling, and transport of pathogenic specimens, as well as molecular biology-based methods for the rapid detection and accurate identification of TB-specific nucleic acids in such specimens are highly desired.